Complete genome sequence of Methanoplanus petrolearius type strain (SEBR 4847T)

Methanoplanus petrolearius Ollivier et al. 1998 is the type strain of the genus Methanoplanus. The strain was originally isolated from an offshore oil field from the Gulf of Guinea. Members of the genus Methanoplanus are of interest because they play an important role in the carbon cycle and also because of their significant contribution to the global warming by methane emission in the atmosphere. Like other archaea of the family Methanomicrobiales, the members of the genus Methanoplanus are able to use CO2 and H2 as a source of carbon and energy; acetate is required for growth and probably also serves as carbon source. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the family Methanomicrobiaceae and the sixth complete genome sequence from the order Methanomicrobiales. The 2,843,290 bp long genome with its 2,824 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain SEBR 4847 T (= DSM 11571 = OCM 486) is the type strain of Methanoplanus petrolearius [1]. This strain was isolated from an offshore oilproducing well in the Gulf of Guinea, Africa [1]. Currently, the genus Methanoplanus contains three species: M. petrolearius, the type species M. limicola (isolated from an Italian swamp containing drilling waste near Baia in the Naples Area), and M. endosymbiosus (isolated from the marine ciliate Metopus contortus) [1]. The genus name derived from the Latin word "methanum", and the adjective "planus", meaning a flat plate, which refers to its flat cell morphology [1,2]. Methanoplanus therefore means "methane (-producing) plate". The species epithet petrolearius derives from the Latin word "petra", rock and the adjective "olearius", which relates to vegetable oil [1]. "Petrolearius" means therefore related to mineral oil, referring to its origin of isolation [1]. No additional cultivated strains belonging to the species M. petrolearius have been described thus far. M. petrolearius SEBR 4847 T is like other methanogens, strictly anaerobic. Here we present a summary classification and a set of features for M. petrolearius strain SEBR 4847 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
The type strains of the two other species in the genus Methanoplanus share an average of 93.5% 16S rRNA gene sequence identity with strain SEBR 4847 T [1,2]. The 16S rRNA gene sequence of the strain SEBR 4847 T shows 99% identity with an uncultured environmental 16S rRNA gene se-quence of the clone KO-Eth-A (AB236050) obtained from the marine sediment [3]. The 16S rRNA gene sequences similarities of the strain SEBR 4847 T to metagenomic libraries (env_nt) were all 83% or less, (status August 2010), indicating that members of the species, genus and even family are poorly represented in the habitats screened thus far. Figure 1 shows the phylogenetic neighborhood of M. petrolearius SEBR 4847 T in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome do not differ from the previously published 16S rRNA sequence generated from DSM 11571 (U76631), which contained four ambiguous base calls.  [4,5] of the 16S rRNA gene sequence under the maximum likelihood criterion [6] and rooted with Methanocellales [7]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 350 bootstrap replicates [8] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [9] are shown in blue, published genomes in bold [10,11] and Gen-Bank accessions CP001338 (for Methanosphaera palustris E1-9c) and AP011532 (for Methanocella paludicola).
The cells of strain SEBR 4847 T stain Gramnegative, but archaea do not have a Gram-negative type of cell wall with an outer envelope. Cells occur singly or in pairs and are irregularly disc-shaped of 1 to 3 µm size ( Figure 2 and Table 1). A similar shape was found for two other strains of the genus Methanoplanus [1,2,24]. Strain SEBR 4847 T was originally described as non-motile [1], however, in samples of this strain kept in the DSMZ culture collection motile cells were frequently detected in young cultures (H. Hippe, personal communication). The genome sequence of SEBR 4847 T contains numerous genes encoding flagellins (Mpet_2052 -Mpet2054, Mpet_2057) and chemotaxis proteins (Mpet_2064 -Mpet_2069), which is in line with the observation of motility in this species. Round colonies of 1-2 mm are observed after three weeks of incubation on solid agar medium. The generation time of strain SEBR 4847 T is about 10 hours under optimal conditions [1]. Strain SEBR 4847 T grows optimally at 37°C, the temperature range for growth being 28-43°C. No growth was observed at 25°C or 45°C [1]. The optimum pH is 7.0; growth occurs from pH 5.3 to 8.4. The optimum NaCl concentration for growth is between 1 and 3% NaCl with growth occurring at NaCl concentrations ranging from 0 to 5% [1]. Substrates for growth of strain SEBR 4847 T are H2 + CO2, formate and CO2 + 2propanol [1]. Strain SEBR 4847 T does not utilize methanol, trimethylamine, lactate, glucose, CO2 + 1-propanol, CO2 + 1-butanol and isobutyrate [1]. Acetate is required for growth as carbon source and yeast extract is stimulatory [1]. Addition of acetate reduces the lag time [25]. The addition of acetate slightly increases the amount of H2 available, theoretically [26,27]. When H2 is limiting and sulfate is in excess, sulfate reducers compete with methanogens and homoacetogens for the available H2 [27]. The sulfate reducers can out-compete hydrogenotrophic methanogens, due to a higher affinity [28] and higher activity of hydrogenase and the energetically more favorable reduction of sulfate [29]. Similar features were observed for M. limicola and M. endosymbiosus [1,2,24].  [2,24]. Preparations of the cell envelope from M. limicola and M. endosymbiosius revealed the presence of a dominant band that appeared to be a glycoprotein when cells were disrupted in 2% SDS [2,24]. Methanoplanus spp. possesses a mixture of C20C20 and C40C40 core ethers [30]. For comparison, similar mixtures were also detected in other members of the family Methanomicrobiaceae: Methanogenium cariaci, Methanogenium marisnigri and Methanogenium thermophilicum, while C20C25 was absent in these species [30]. Standards in Genomic Sciences , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [23]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [31], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [32]. The genome project is deposited in the Genome OnLine Database [9] and the com-plete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website. Pyrosequencing reads were assembled using the Newbler assembler Version 2.3 Pre-Release-09-14-2009 (Roche). The initial Newbler assembly consisted of 21 contigs in one scaffold that was converted into a phrap assembly by making fake reads from the consensus sequence. Illumina GAii sequencing data (148.5Mb) was assembled with Velvet [34] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The draft assembly was based on 173.4 Mb of 454 data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment of the genome sequence. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution, Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [35]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 139 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [36]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 120.1× coverage of the genome. The final assembly of the genoe contains 590,575 pyrosequences and 4,125,153 Illumina reads.

Genome annotation
Genes were identified using Prodigal [37] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [38]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uni-Prot, TIGRFam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [39].

Genome properties
The genome consists of a 2,843,290 bp long chromosome with a 47.4% GC content (Table 3 and Figure 3). Of the 2,881 genes predicted, 2,825 were protein-coding genes, and 57 RNAs; thirty nine pseudogenes were also identified. The majority of the protein-coding genes (61.2%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.